The purpose of this research is to understand how TIMP (tissue inhibitor of metalloproteinases) accomplishes its anticarcinogenic action in immortal 3T3 cells. To do this, we propose to perform genetic surgery on the mouse cDNA sequence encoding murine TIMP, using recombinant DNA techniques, to make site-directed mutations in the "mini-gene". The mutant TIMP proteins will be produced in E. coli and their ability to inhibit the known matrix metalloproteinases (MMPs) assessed. These studies will tell us which portions of the protein are important to interact with the MMPS, and they will yield penetrating insights into the differences in the active sites of the various MMPs (which exhibit different substrate specificities). our experiments will proceed along two parallel tracks, one (spec. aim #3) concerned with understanding the details of TIMP structure, the other (spec. aim #1) dealing with developing mutant TIMPS able to discriminate among the MMPS. The second specific aim is to make minigenes encoding TIMPS with inhibitory activity against selected MMPs and to transfect them, as part of an expression cassette, into tumorigenic, metastatic cells (both our TIMP down-modulated lines and appropriate human lines) to determine their ability to suppress the oncogenic phenotype. The significance of this research lies in the knowledge it will produce regarding how TIMP functions in vivo to suppress tumor development. Is an MMP a target, and if so, which one is it? A better understanding of the structure of TIMP and how it interacts with the MMPs will be of fundamental importance not only to cancer but also to diseases of the joints (arthritis) and connective tissue pathologies. Although direct evidence that TIMP has the properties of an "anti-oncogene" in human cancers is lacking, it is hard to believe that it does not have a significant role in suppressing or hindering the development of a malignancy and moderating its invasiveness.